Ca2+ ions function as intracellular second messengers that regulate a wide variety of cellular activities including fertilization, apoptosis, muscle contraction, lymphocyte activation, and, most pertinent to this proposal, cell cycle control and proliferation. Precise control of the cell cycle is critical throughout development, and Ca2+ functions in signal transduction pathways that regulate the cell cycle as early as fertilization. Therefore, delineation of the Ca2+ signaling mechanisms that regulate the cell cycle is paramount to our understanding of developmental processes. A clear understanding of the basic mechanisms that regulate the cell cycle is also fundamental to our ability to prevent and treat cancer, which is caused by abnormal cell cycle regulation. The long-range goals of this project are to understand the basic mechanisms by which Ca2+ signaling regulates the cell cycle and proliferation, particularly during development, and to identify Ca2+ regulatory mechanisms that may be potential therapeutic targets in the prevention and treatment of cancer. In the past year, we have focussing on understanding how ER morphology and arrangement influences its function. The ER undergoes significant reorganization between interphase and mitosis, but the underlying mechanisms are unknown. Stromal interaction molecule 1 (STIM1) is an ER Ca2+ sensor that activates store-operated Ca2+ entry (SOCE) and also functions in ER morphogenesis through its interaction with the microtubule +TIP protein end binding 1 (EB1). We previously demonstrated that phosphorylation of STIM1 during mitosis suppresses SOCE. We now show that STIM1 phosphorylation is a major regulatory mechanism that excludes ER from the mitotic spindle. In mitotic HeLa cells, the ER forms concentric sheets largely excluded from the mitotic spindle. We show that STIM1 dissociates from EB1 in mitosis and localizes to the concentric ER sheets. However, a nonphosphorylatable STIM1 mutant (STIM110A) colocalized extensively with EB1 and drove ER mislocalization by pulling ER tubules into the spindle. This effect was rescued by mutating the EB1 interaction site of STIM110A, demonstrating that aberrant association of STIM110A with EB1 is responsible for the ER mislocalization. A STIM1 phosphomimetic exhibited significantly impaired +TIP tracking in interphase, but was ineffective at inhibiting SOCE, suggesting different mechanisms of regulation of these two STIM1 functions by phosphorylation. Thus, ER spindle exclusion and ER-dependent Ca2+ signaling during mitosis require multimodal STIM1 regulation by phosphorylation. In conclution, remodeling of the EB1-associated complex of +TIPS may be necessary during mitosis, and the functional significance of this remodeling requires significant future investigation.